CN109342743B - Preparation method of denatured IgG capable of being efficiently combined with rheumatoid factor - Google Patents

Preparation method of denatured IgG capable of being efficiently combined with rheumatoid factor Download PDF

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CN109342743B
CN109342743B CN201811482681.4A CN201811482681A CN109342743B CN 109342743 B CN109342743 B CN 109342743B CN 201811482681 A CN201811482681 A CN 201811482681A CN 109342743 B CN109342743 B CN 109342743B
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CN109342743A (en
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柯拓
曾家骅
岑静
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Fapon Biotech Inc
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Abstract

The invention relates to the field of medical diagnosis, in particular to a preparation method of denatured IgG capable of being efficiently combined with rheumatoid factors. The method comprises the following steps: a) denaturing the IgG, and b) polymerizing the denatured IgG using a protein cross-linking agent. The denatured IgG prepared by the method has high reaction activity with rheumatoid factors, wider detection range and better accuracy, and has better clinical application value.

Description

Preparation method of denatured IgG capable of being efficiently combined with rheumatoid factor
Technical Field
The invention relates to the field of medical diagnosis, in particular to a preparation method of denatured IgG capable of being efficiently combined with rheumatoid factors.
Background
Rheumatoid Arthritis (RA) is a common rheumatic disease, joint deformity often appears 1-3 years after the disease starts, daily work and life are seriously affected, and the problem concerned by medical workers is that the early diagnosis rate is improved. Rheumatoid Factor (RF) is one of the important criteria for diagnosing rheumatoid arthritis, and it is very important to quickly, efficiently and accurately detect RF for the diagnosis and prognosis of RA.
RF is an autoantibody produced by abnormal IgG in the human body which stimulates the body, and mainly belongs to the IgM class. In the in vitro diagnosis of RF, denatured IgG is indispensable, and its binding ability to RF is an important guarantee for the accuracy of RF detection and the precision of diagnosis.
In the prior art, the conventional denatured IgG is prepared by extracting normal IgG, and then performing thermal denaturation at 63 ℃ for 30min, or treating 6M urea and 0.1M beta-mercaptoethanol at 37 ℃ and the like.
Denatured IgG prepared by these methods has limited binding ability to RF and poor performance in clinical applications. And the heat treatment process is difficult to control accurately, and the batch-to-batch difference fluctuation of the sample after thermal denaturation is large.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a preparation method of denatured IgG, and the denatured IgG prepared by the method has high reactivity with Rheumatoid Factor (RF), wider detection range, better accuracy and better clinical application value.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the first aspect of the invention relates to a preparation method of denatured IgG capable of efficiently binding with rheumatoid factor, which comprises the following steps:
a) denaturing the IgG, and
b) denatured IgG was polymerized using a protein cross-linker.
According to a second aspect of the invention, the invention also relates to a rheumatoid factor detection kit, which comprises a solid phase carrier coated with the denatured IgG prepared by the method.
Compared with the prior art, the method changes the denaturation means of normal IgG on the basis of the background technology, prepares the denatured IgG efficiently combined with RF, and can effectively improve the performance of the RF detection reagent in clinical application. The denatured IgG prepared by the method provided by the invention has strong binding capacity with RF, high reactivity, wide detection range in clinical biochemical detection, high accuracy and excellent performance.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the response of one example of the present invention and a comparative example when RF detection is performed.
Detailed Description
The invention relates to a preparation method of denatured IgG capable of being efficiently combined with rheumatoid factors, which comprises the following steps:
a) denaturing the IgG, and
b) denatured IgG was polymerized using a protein cross-linker.
In some embodiments, the protein cross-linking agent is selected from glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), 4- (N-maleimidomethyl) cyclohexanecarboxylic acid-N-succinimidyl ester (SMCC), Disuccinimidyl sulfate (DSS), DST, 3- [ (2-aminoethyl) dithio ] propionic Acid (AEDP), MBS, N' -dicyclohexylcarbodiimide (dcc), SPDP, EGS, Transglutaminase (TG), Peroxidase (POD), and polyphenol oxidase (PPO).
In some embodiments, the protein crosslinking agent is preferably one whose directing groups are amino and carboxyl groups, such as EDC, AEDP, and DCC.
In some embodiments, the protein cross-linking agent is EDC, and the mass ratio of EDC to denatured IgG when subjected to the polymerization treatment is (2-3): 1.
in some embodiments, the protein cross-linking agent is EDC, and the mass ratio of EDC to denatured IgG when subjected to the polymerization process is 2.5: 1.
in some embodiments, the sample to be tested is selected from the group consisting of cell culture supernatant, whole blood, plasma, serum, tissue, or tissue lysate.
As used herein, "tissue lysate," "cell lysate," "lysed sample," "tissue extract," or "cell extract" refers to a sample and/or biological sample material comprising lysed tissue or cells, i.e., wherein the structural integrity of the tissue or cells has been disrupted. To release the contents of a cell or tissue sample, the material is typically treated with enzymes and/or chemical agents to lyse, degrade, or disrupt the cell walls and membranes of such tissues or cells. The skilled artisan is well familiar with suitable methods for obtaining a lysate. This process is encompassed by the term "lysis".
In some embodiments, the IgG is of a species selected from cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fighting, or human.
In some embodiments, the IgG is subjected to a denaturation treatment selected from the group consisting of heat treatment, acid treatment, alkali treatment, chemical modification, disruption of hydrogen bonds of the protein, disruption of hydrophobic structure of the protein, oxidation treatment, and enzymatic cleavage treatment.
In some embodiments, the IgG is denatured by digestion with papain or trypsin.
In some embodiments, the IgG is denatured by treating at 60-66 ℃ for 20-40 min.
In some embodiments, the IgG is denatured using one or more of SDS, urea, or beta mercaptoethanol.
The denaturation treatment by SDS, urea or beta mercaptoethanol can avoid the defect of large batch-to-batch difference fluctuation of the heat treatment process, and the operation is simple and convenient. This denaturation method works well with subsequent protein cross-linker polymerization to improve the ability of IgG to react with RF.
In some embodiments, the IgG is denatured by 0.08-0.12 w/v% SDS at 35-39 ℃ for 1.5-2.5 h.
In some embodiments, the IgG is denatured by 0.015-0.025 w/v% SDS at 35-39 deg.C for 2-3 h.
In some embodiments, the IgG is denatured by 5M to 7M urea, 0.08M to 0.12M beta mercaptoethanol, and treatment at 35 ℃ to 39 ℃ for 1.5h to 2.5 h.
In some embodiments, before step a), or after step a) and before b), further comprising: and (4) enriching IgG.
In some embodiments, the method for enriching IgG is selected from the group consisting of ProteinA affinity purification, ion exchange chromatography (DEAE column chromatography), saturated ammonium sulfate precipitation, organic solvent precipitation (ethanol, caprylic acid), PEG displacement, polyamide composite membrane affinity membrane method.
In some embodiments, the ion exchange chromatography is DEAE column chromatography.
In some embodiments, the organic solvent precipitation method is selected from ethanol precipitation or caprylic acid precipitation.
According to another aspect of the present invention, the present invention also relates to a rheumatoid factor detection kit comprising a solid support coated with denatured IgG prepared as described above.
In some embodiments, the solid support is a microplate, a polystyrene latex particle, a dipstick, or a magnetic nanoparticle.
Denatured IgG is coated onto polystyrene latex particles, and this sensitizing latex undergoes gross agglutination when it encounters RF in the sample being tested.
In some embodiments, the microwell plate is a polystyrene plate; if the sample to be detected contains RF, the RF can be combined with the sample and reacts with the enzyme labeled thermal agglutination IgG which is added subsequently, and finally the substrate is added for coloration, and the RF level can be judged according to the coloration degree.
In some embodiments, the denatured IgG is labeled with an indicator for indicating signal intensity.
In some embodiments, the indicator that shows signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxigenin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron-dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.
In some embodiments, the fluorescent species include Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein, 5-carboxy-2 ', 4', 5 ', 7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate, diamine, bispyanin, La Jolla Blue dye, allophycocyanin, allocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and texas red.
In some embodiments, the radioisotope comprises110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154-158Gd、32P、11C、13N、15O、186Re、188Re、51Mn、52mMn、55Co、72As、75Br、76Br、82mRb and83sr.
In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.
In some embodiments, the fluorescent microspheres are: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.
According to one aspect of the invention, the invention also relates to a method for detecting rheumatoid factor (or diagnosis of rheumatoid arthritis), said method comprising:
using polystyrene latex particles coated with the denatured IgG prepared by the method to contact with a sample to be detected, and indicating the existence of rheumatoid factors if macroscopic agglutination occurs;
or;
the denatured IgG prepared by the method described above is labeled with an indicator for indicating signal intensity, and the denatured IgG is contacted with a detection rheumatoid factor to produce an immune complex, and the presence of the detection rheumatoid factor is evaluated by detecting the intensity of the indicator; alternatively, in the above detection process, the formation of the immune complex may also include the participation of denatured IgG of unlabeled indicator (e.g., double antibody sandwich method), or anti-human IgG antibody (e.g., indirect ELISA method);
in a preferred embodiment, the anti-human IgG antibody is selected from the group consisting of F (ab')2And (3) fragment.
An ideal scenario for diagnosis is a situation where a single event or process may cause various diseases, e.g. in infectious diseases. In all other cases, correct diagnosis can be very difficult, especially when the etiology of the disease is not fully understood. As the skilled artisan will appreciate, a diagnosis without biochemical markers is 100% specific and with 100% sensitivity. Conversely, detection of rheumatoid factor in combination with other clinically usual clinical indications can be used to assess the presence or absence or severity of rheumatoid arthritis with some likelihood or predictive value. Thus, in routine clinical diagnosis, a combination of various clinical symptoms and biological markers is often considered to diagnose, treat and control underlying diseases.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
Extraction of mouse IgG
20mL of the serum from the negative mouse was collected, and an equal volume of saturated ammonium sulfate was added to the serum at 50% saturation with stirring. Continuously stirring for 5min, standing at 4 ℃ for 1h, and precipitating immunoglobulin;
centrifuging at 12000rpm for 10min, dissolving precipitate with 500ml of 1xPBS (10mM PB +150mM NaCl, pH7.4), dialyzing to 10L PBS, and changing the solution once after overnight;
centrifuging at 12000rpm for 10min, and collecting the supernatant. The supernatant was eluted through ProteinG, 100mM Gly PH2.7, and the eluate was collected as pure human IgG and dialyzed into PBS.
The sample is centrifuged, and the supernatant is the human IgG sample to be denatured.
Second, denaturation treatment of IgG
Diluting the sample to 10mg/ml with PBS, and treating at 63 ℃ for 30 min;
putting the sample to room temperature, and adding glutaraldehyde for crosslinking;
the samples were dialyzed, replaced in a 1XPBS system and supplemented with 0.09% NaN3The product is denatured IgG.
Example 2
Extraction of human IgG
Purifying by polyamide composite membrane affinity membrane method.
Preparing 20 affinity membranes with 47mm diameter and phenylalanine ligand by trichlorotriazine bonding method, assembling in a disc-type affinity membrane separator, and separating with 0.05mol/L NaH2PO4Diluting 4mL of human plasma by the solution, pumping the diluted plasma liquid into a human affinity membrane separator at the flow rate of 1mL/min, eluting residual foreign proteins on the membrane by using 1mol/L of NaCl solution 50mI. at the flow rate of 2mL/min, eluting IgG subjected to affinity adsorption on the membrane at the same flow rate by using 1mol/L of sodium chloride/ethylene glycol (equal volume), collecting eluent, dialyzing and collecting a sample.
Second, denaturation treatment of IgG
Diluting the sample to 10mg/ml with PBS, treating the sample with 6M urea and 0.1M beta mercaptoethanol at 37 ℃ for 2 h;
the sample is placed to room temperature, and AEDP is added for crosslinking;
the samples were dialyzed, replaced in a 1XPBS system and supplemented with 0.09% NaN3The product is denatured IgG.
Example 3
Extraction of human IgG
1L of negative human serum was collected, and an equal volume of saturated ammonium sulfate was added to the collected serum at 50% saturation with stirring. Continuously stirring for 5min, standing at 4 ℃ for 1h, and precipitating immunoglobulin;
centrifuging at 12000rpm for 10min, dissolving precipitate with 500ml of 1xPBS (10mM PB +150mM NaCl, pH7.4), dialyzing to 10L PBS, and changing the solution once after overnight;
centrifuging at 12000rpm for 10min, and collecting the supernatant. The supernatant was eluted through ProteinG, 100mM Gly PH2.7, and the eluate was collected as pure human IgG and dialyzed into PBS.
The sample is centrifuged, and the supernatant is the human IgG sample to be denatured.
Second, denaturation treatment of IgG
Diluting the sample to 10mg/ml with PBS, adding SDS (w/v) according to 0.02%, and treating for 2.5h in 37-degree water bath;
placing the sample at room temperature, adding DCC solid with 2.5 times of protein mass (EDC: protein amount is 2.5:1), mixing, and standing at room temperature for 1 h;
the samples were dialyzed, replaced in a 1XPBS system and supplemented with 0.09% NaN3The product is denatured IgG.
Example 4
Extraction of human IgG
Purifying by polyamide composite membrane affinity membrane method.
Adding equal amount of 0.025M Phosphate Buffer Solution (PBS) with pH7.8 into 10mL human serum, adding PEG solution with molecular weight of 1000 to the final concentration of 16%, mixing the samples on a vortex mixer, placing the mixture in a refrigerator (4C) overnight, centrifuging for 5-10 min (2500 rpm) the next day, discarding the supernatant, dissolving the precipitate with 0.025M PBS with pH7.8, performing DEAE cellulose column chromatography directly, and eluting with 0.0175M PBS with pH 6.3.
Second, denaturation treatment of IgG
The samples were diluted to 10mg/ml with PBS, 0.1 w/v% SDS, treated for 2h at 37 ℃. (ii) a
Putting the sample at room temperature, and adding DCC for crosslinking;
the samples were dialyzed, replaced in a 1XPBS system and supplemented with 0.09% NaN3The product is denatured IgG.
Comparative example 1
Extraction of human IgG
The same as in example 3.
Second, denaturation treatment of IgG
Diluting the sample to 10mg/ml with PBS, adding SDS (w/v) according to 0.02%, and treating for 2.5h in 37-degree water bath;
the samples were dialyzed, replaced in a 1XPBS system and supplemented with 0.09% NaN3The product is denatured IgG.
The denatured IgG prepared in example 3 and comparative example 1 was used for RF detection by a biochemical platform latex enhanced immunoturbidimetry:
1. coupling denatured IgG to activated carboxyl microspheres, R2;
2. diluting the calibrated RF sample in a gradient manner to prepare a calibration point S;
3. performing biochemical test, namely detecting R1SR2 on a biochemical platform to obtain a series of reaction degrees;
4. the concentration of the calibration points is plotted against the degree of reaction.
The results are shown in FIG. 1 (the abscissa is RF activity concentration in IU/mL, and the ordinate is reactivity), from which it can be seen that: the denatured IgG prepared in example 3 was superior to the comparative examples in terms of reactivity, linear range, etc.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (5)

1. A method for preparing denatured IgG capable of efficiently binding to rheumatoid factor, comprising:
a) denaturing the IgG, and
b) polymerizing the denatured IgG using a protein cross-linking agent;
the denaturant used for the denaturation treatment of the IgG is 0.08 w/v% -0.12 w/v% SDS; the treatment condition is that the treatment is carried out for 1.5 to 2.5 hours at the temperature of between 35 and 39 ℃;
the protein cross-linking agent is EDC, and when polymerization treatment is carried out, the mass ratio of the EDC to the denatured IgG is (2-3): 1;
the preparation method of the denatured IgG is used for detecting the rheumatoid factor by a latex enhanced immunoturbidimetry method; the sample to be tested is selected from cell culture supernatant, whole blood, plasma, serum, tissue or tissue lysate.
2. The method of claim 1, wherein the IgG species is selected from the group consisting of bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, and human.
3. The method according to claim 1, wherein before step a), or after step a), and before b), further comprising: and (4) enriching IgG.
4. The method of claim 3, wherein the IgG is enriched by a method selected from the group consisting of ProteinA affinity purification, ion exchange chromatography, ammonium sulfate saturation precipitation, organic solvent precipitation, PEG displacement, and polyamide composite membrane affinity membrane.
5. A kit for detecting rheumatoid factor latex by enhanced immunoturbidimetry, which comprises polypropylene latex particles coated with denatured IgG prepared by the method of any one of claims 1 to 4.
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